Project Summary: Naturally arising CD4+ CD25+ Foxp3+ regulatory T (Treg) cells play an important role in the maintenance of immune homeostasis and prevention of autoimmunity. It has long been known that the majority of Treg cells develop in the thymus, yet it has become clear that Treg cells can also develop in the periphery. However, the cellular and molecular mechanisms by which self-reactive thymocytes or peripheral T cells are directed to express Foxp3 in vivo remain unclear. We have begun to address these issues by dissecting the developmental stages involved in thymic Treg cell development. We found that Foxp3+ CD25hi CD4+ CD8- thymocytes are enriched in cells which develop into Foxp3+ Treg cells after intrathymic transfer, suggesting the existence of an intermediate in thymic Treg cell development. Our goal is to characterize this thymic Treg cell precursor using cellular and molecular approaches, and utilize this information in our evaluation of TCR and CD28 signals in the process of thymic Treg cell development (Aim 1). We have also begun to evaluate the mechanisms of peripheral Treg cell development (Aim 2), and have found that TCR specificity plays a critical role in the generation of Foxp3+ cell in the periphery. We also plan to leverage our studies of thymic Treg cell precursors and ask whether these mechanisms also apply to the peripheral development of Treg cells. We believe that our overall goal of understanding the mechanisms that govern Treg cell development may lead to the development of therapies which induce or inhibit development of Treg cells for treatment of autoimmunity, transplantation, or cancer. Public Health Relevance: Regulatory T cells are a subset of immune cells which are potent inhibitors of immune inflammation. Their normal role is to prevent autoimmune disease, but can also prevent rejection during transplantation. On the other hand, regulatory T cells can also inhibit the immune responses to tumors, decreasing the efficacy of cancer immunotherapy. Our goal is to understand the mechanisms by which regulatory T cell develop, with the notion that this information can help us design therapies which enhance or diminish the generation of regulatory T cells so that the immune system will become more or less aggressive, facilitating treatment of diseases such as cancer or autoimmunity, respectively.